The Journal of Immunology Mast Cell–deficient KitW-sh ‘‘Sash’’ Mutant Mice Display Aberrant Myelopoiesis Leading to the Accumulation of Splenocytes That Act as Myeloid-Derived Suppressor Cells Anastasija Michel,* Andrea Schu¨ler,† Pamela Friedrich,* Fatma Do¨ner,* Tobias Bopp,* Markus Radsak,† Markus Hoffmann,* Manfred Relle,‡ Ute Distler,* Jo¨rg Kuharev,* Stefan Tenzer,* Thorsten B. Feyerabend,x Hans-Reimer Rodewald,x Hansjo¨rg Schild,* Edgar Schmitt,* Marc Becker,‡ and Michael Stassen* Mast cell-deficient KitW-sh ‘‘sash’’ mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that KitW-sh causes aberrant extramedul- lary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor- bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell–independent phenomenon. Thus, the KitW-sh mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research. The Journal of Immunology, 2013, 190: 5534–5544. he receptor tyrosine kinase c-Kit (CD117) and its ligand rocytic anemia, sterility, pigmentation defects, and intestinal dis- stem cell factor (SCF) have been intensively studied owing orders (7). T to their multifaceted role in development and hemato- The c-Kit/SCF axis is also crucial for the development of mast poiesis (1, 2). These molecules were mapped to the White spotting cells, a finding that led to the widespread use of several strains of c- and Steel loci, respectively, and a variety of mutant alleles have Kit/SCF mutant mice in mast cell research (8). The composite been described (3–6). genotype KitW/KitW-v causes severe mast cell deficiency (9), yet Mice carrying spontaneous loss-of-function mutations at either these animals are also anemic and infertile. More recently, mast Steel or White spotting loci generally show pleiotropic yet similar cell–deficient KitW-sh/KitW-sh ‘‘sash’’ mice serve as a common phenotypes. Pathophysiological manifestations can include mac- tool, as these animals do not suffer from some disadvantages that characterize the KitW/KitW-v strain (10). Although sash mice also lack mast cells, melanocytes, and interstitial cells of Cajal, they *Institute for Immunology, University Medical Center of the Johannes Gutenberg are fertile, not anemic, and show normal numbers of gd T cells University, 55131 Mainz, Germany; †III Medical Department, University Medical ‡ (11–13). Center of the Johannes Gutenberg University, 55131 Mainz, Germany; I Medical W-sh Department, University Medical Center of the Johannes Gutenberg University, The Kit mutation is an inversion of ∼3.1 Mbp encompass- x 55131 Mainz, Germany; and Division for Cellular Immunology, German Cancer ing 27 known genes. However, in contrast to the KitW and KitW-v Research Center, D-69120 Heidelberg, Germany mutations, KitW-sh does not alter the coding region of White spotting Received for publication December 6, 2012. Accepted for publication March 29, locus itself. The 59 breakpoint of this inversion disrupts the Corin 2013. gene, leading to cardiac hypertrophy, and the 39 breakpoint is lo- This work was supported by Deutsche Forschungsgemeinschaft Grants STA 984/4-1 (to M.S. and M. Radsak), STA 984/1-2 (to M.S.), and Ra 988/4-2 (to M. Radsak and cated72kbpupstreamofthec-Kit transcriptional start site (14, H.S.) the European Research Council (Advanced Grant 233074, to H.-R.R.), and by 15). Regulatory elements driving the expression of c-Kit in mast the Immunology Center of Excellence Mainz (to M.S.). cells were mapped within the affected region (16). Thus, it can be Address correspondence and reprint requests to Dr. Michael Stassen, Institute for assumed that the inversion or additional deletions of cis regulatory Immunology, University Medical Center of the Johannes Gutenberg University, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail address: [email protected] elements prevent mast cell–specific c-Kit expression, which ulti- The online version of this article contains supplemental material. mately leads to their irreversible demise within a few weeks after birth of sash mice (17, 18). Abbreviations used in this article: CMP, common myeloid progenitor; G-MDSC, granulocytic myeloid-derived suppressor cell; HSC, hematopoietic stem cell; L1C2, The use of Kit mutant mice has been of central importance to G line 1 alveolar cell carcinoma; Lin, lineage; L-NMMA, N -monomethyl-L-arginine; unravel mast cell functions in the immune system and beyond (19, LSK, Lin2Sca-1+c-Kit+; LT-HSC, long-term hematopoietic stem cell; MDSC, myeloid-derived suppressor cell; MEP, megakaryotic/erythroid progenitor; M-MDSC, 20). However, novel mast cell–deficient strains with unperturbed monocytic myeloid-derived suppressor cell; MPP, multipotent progenitor; MS, mass c-Kit/SCF function challenge some findings obtained in Kit mu- v spectrometry; nor-NOHA, N -hydroxy-nor-L-arginine; ROS, reactive oxygen spe- tant mice (21, 22). These inconsistent observations may indicate cies; SCF, stem cell factor; ST-HSC, short-term hematopoietic stem cell. limitations of mast cell–dependent mouse strains with mutant Kit Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 alleles (23–25). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203355 The Journal of Immunology 5535 It this study, we demonstrate that the KitW-sh defect causes ex- and cells were stained for CD11b, Ly6G, Ly6C, or only with propidium io- tramedullary hematopoiesis leading to the accumulation of mye- dide and phenotype was analyzed via flow cytometry. + + loid progenitor cells in the spleen of naive sash mice. CD11b Ly6C Histology cells that express intermediate and high levels of Ly6G are also highly expanded in sash spleen. Morphologically and functionally, Cytospin preparations were stained with a microscopy Hemacolor set (Merck) according to the manufacturer’s recommendations. Slides were these Ly6G-expressing cells resemble granulocytic myeloid-derived analyzed by bright-field microscopy on a Keyence BZ-8000 fluorescence suppressor cells (G-MDSC), which typically expand in tumor- microscope. bearing hosts. Thus, besides its well-documented roles on mast To assess mast cell numbers, ears were removed, fixed in Roti-Histofix cell development and survival, the KitW-sh allele affects myelopoi- (Roth), and embedded in paraffin. Sections were deparaffinized, rehydrated, and stained with avidin-Alexa Fluor 488 (Invitrogen). Slides were analyzed esis, which may have adverse effects regarding the interpretation of in GFP channel on a Keyence BZ-8000 fluorescence microscope. mast cell–dependent phenomena. Bone marrow chimeras Materials and Methods BALB/c mice were lethally irradiated with 8.5 Gy from a [137Cs] source Mice (OB58-BA; Buchler Braunschweig). Next day, 5 3 106 bone marrow donor cells from C.B6-KitW-sh or from BALB/c mice were transferred by W-sh d C.B6-Kit mice (H-2 ) were generated as previously described and i.v. injection. Then, mice were housed under specific pathogen-free con- backcrossed at least 12 generations (26). BALB/c wild-type littermates ditions for a time period of 8 wk before use. were obtained from crosses between heterozygous C.B6-KitW-sh/+ mice. Cpa3Cre/+ mice (22) on a BALB/c background and their wild-type litter- Genotyping mates were obtained from Thorsten Feyerabend (German Cancer Research Center). Cells sorted by flow cytometry cells were genotyped according to a pub- All mice were used in accordance with the guidelines of the Central lished procedure (15). Animal Facility of the University of Mainz. Generation of BMDC Abs and reagents Single bone marrow cell suspensions were cultured in IMDM supple- CD11b-FITC (M1/70), Ly6G-PE (1A8), CD11b-biotin (M1/70), Ly6C- mented with 5% FCS, 1 mM L-glutamine, 1 mM NaPyr, and 50 ng/ml GM- CSF. Medium was changed on days 2 and 4. BMDC were used at day 6 and PerCP-Cy5.5 (HK1.4), CD135-PE (A2F10), CD117-PE-Cy7 (ACK2), + CD16/32 (93) , CD34-FITC (RAM34), and CD150-FITC (9D1) were ob- were at least 85% CD11c . tained from eBioscience. Ly6G-FITC (1A8), CD117-allophycocyanin Allogeneic MLR (ACK2), Sca-1-allophycocyanin (D7), CD8a-biotin (53-6.7), CD5-biotin (53-7.3), CD45R (B220)-biotin (RA3-6B2), CD127-biotin (A7R34), TER- To explore the suppressive capacity of CD11b+ subpopulations on T cells, 119-biotin, Gr-1-biotin (RB6-8C5), and streptavidin-PerCP were obtained sorted cells were given to lymphocytes in a ratio of 1:1, 1:3, or 1:9. from BioLegend. CD16/32-PE (2.4G2) and CD3ε-biotin (500A2) were In general, the allogeneic MLR consisted of 5 3 104 lymphocytes from obtained from BD Biosciences. Propidium iodide was from Sigma- BALB/c mice as responders and 1.67 3 103 BMDC from C57BL/6 mice Aldrich, and CD4-biotin (H129.19) was by our own production and bio- as activators. The ratio of BMDC to lymphocytes (1:30) was constant in all tinylation. experiments. On day 4, [3H]thymidine (0.5 mCi/well) was added and the incorporation was determined in a liquid scintillation counter (LKB Flow cytometry analyses and cell sorting Wallac) 20 h later.